The function of the thymus gland, which lies just beneath the breast bone, was only first revealed in 1960. Prior to that time, the thymus was thought to be of little importance since in adults it is almost non-existent because of rapid atrophy after adolescence. As was the case with other organs (e.g., the pancreas), the function of the thymus was suggested by observing the effect of its removal in young animals. When pre-adolescent animals are thymectomized, they experience a profound "wasting disease" which is characterized by a variety of maladies, including increased incidence of infection and cancer, failure to grow, allergies and neuromuscular paralysis. The greater susceptibility to infection and cancer was shown to be directly attributable to a dramatic decrease in peripheral blood lymphocytes, and could be prevented by rearing the animal in a germ-free environment. However, the other symptoms of thymectomy were not completed abrogated by this approach.
In 1964, it was demonstrated that hormone-like factors from thymus tissue could prevent many of the manifestations of "wasting disease," thus suggesting that the thymus produces substances important in the development of immunity. The relationship of this observation to the other "wasting disease" symptoms was not well understood at that time, however.
B and T lymphocytes are the primary effector cells of the immune response. Both cell classes are considered to derive ultimately from hematopoietic stem cells in the mammalian bone marrow, via progenitor or precursor cells representing distinguishable stages in the differentiation of each class. B lymphocytes, or B cells, are the precursors of circulating antibody-secreting plasma cells. Mature B cells, which are characterized by expression of surface-bound immunoglobulin capable of binding specific antigens, derive from hematopoietic stem cells via an intermediary cell class known as pre-B cells. Mature T cells develop principally in the thymus, presumably from an as yet unidentified precursor cell which migrates from the bone marrow to the thymus at an early stage of T lymphocyte development.
It was not until 1971 that it was discovered that the thymus-derived lymphocytes (T cells) regulated the reactivity of bone marrow-derived antibody-producing lymphocytes (B cells). The latter are involved in the pathogenesis of many autoimmune-type diseases, i.e., those involving the body's reactivity to its own cells or tissues. Examples of such diseases include arthritis, multiple sclerosis, muscular dystrophy, lupus erythematosus, and quite possibly juvenile onset diabetes. Many of the problems associated with "wasting disease" in thymectomized animals are similar to "autoimmune-type" disease. In general, when the thymus gland fails to function properly, T cells, which control the immune response, are defective or absent and the system breaks down.
After the discovery that the thymus was producing a hormone-like factor, several groups of scientists began trying to extract and purify the material from thymus glands, or from serum, in much the same manner that insulin was prepared for therapeutic use in diabetes. The difficulty is that the thymus produces very small quantities of the hormone or hormones. Thus, one requires large amounts of calf thymus or several liters of serum to biochemically extract small amounts of active material. Success has been very limited with this approach.
Very little is known about regulatory factors involved in B and T cell lymphogenesis. In particular, all the factors or conditions required for commitment and expansion of the B and T cell lineages have not yet been defined, albeit it is now known that one or more thymic factors or hormones are produced by the epithelial cells of the thymus gland. (See, e.g., Waksal, et al., Ann. N.Y. Acad. Sci. 249: 493 (1975).) While the ideal approach for studying these factors or hormones would be to isolate these cells from fresh thymic tissue and grow them in vitro, epithelial cells have been extremely difficult to maintain in continuous culture in the laboratory.
Recently, this technical barrier has been overcome, with the presently-disclosed establishment of cloned lines of thymic epithelial cells of feline, canine, bovine and human origin. Earlier efforts to establish a cloned cell line of murine origin laid some of the procedural groundwork for the present invention. (See Beardsley, et al., PNAS 80: 6005 (1983), and Hays & Beardsley, Clin. Immunol. Immunopath. 33: 381 (1984), which are incorporated herein by reference). The present disclosure demonstrates that the thymic epithelial cell lines of the present invention are, in fact, producing factors having activity similar but not identical to substances previously obtained by the difficult and labor-intensive procedure of extracting thymic substances from calf thymus; however, the presently-disclosed factor is in a much purer and more homogeneous form, is produced in greater quantities and has a different and distinct mechanism of action.
The primary activity of the cloned thymic epithelial cell factors produced according to the presently disclosed method has been shown to have the capacity to augment the immune responses of both immature and mature T cells. This factor, which shall be referred to herein as T4 immune stimulating factor ("TISF"), is being further purified and characterized biochemically. Also, in vivo studies have been initiated to determine the efficacy of TISF in enhancing the response of animals to infectious agents and to malignant cells.
Although much of the work done to date has focused on the murine and canine models, the implications and applications to other animals, e.g., felines, bovine species, and especially to humans, have been shown to be equally relevant. As disclosed herein, the difficult step of establishing a human epithelial cell line has also been accomplished successfully. One immediate goal is to apply the various aspects of the present invention, especially TISF, to produce an effective immune potentiator and/or therapeutic agent for the treatment of immunologically-related disease and to produce agents useful in immunization against etiologic agents of disease.